Rhodopsin has recently been recognized as G-protein activating receptor, designed such that the absorption of a photon activates rhodopsin, initiating a sequence of transduction events which lead to the hyperpolarization of the ROS plasma membrane. These events include an amplified activation of G-protein by photoactivated rhodopin, with subsequent activation of a cGMP specific PDE, resulting in closure of cGMP gated sodium channels. The specific aims of the current application are to further characterize the molecular mechanism of regulation of this excitatory cascade by rhodopsin phosphorylation and its subsequent interaction with 48k protein. The energetic coupling of the two defined functional domains of rhodopsin, the retinal binding site and the G-protein interaction site, will be studied to better understand the intramolecular information flow within receptor molecules. The role of the unusual lipid microenvironment of the disk membrane in controlling the functional activation of rhodopsin will be examined. The effect of calcium on the level of light stimulated phosphodiesterase activity and its implication relative to light adaptation will be studied further. The above goals will be accomplished by characterizing a variety of physical and functional properties of purified preparations of rhodopsin molecules, which have been modified by proteolysis, having variable levels of phosphorylation, and amine group modification, reconstituted into lipid bilayers of defined lipid composition. The binding and activation of G-protein and the resulting PDE activation levels, the thermostability, the metaI to metaII equilibrium are among the properties to be studied. The goals of this proposal are to determine the ability of changes in lipid composition, due either to lipid peroxidation or metabolic defects in lipid metabolism, to effect visual pigment function. In addition, the mechanism whereby phosphorylation, which is a major regulatory mechanism in biological control, exerts its regulatory effect will be characterized at the molecular level. The additional role of the 48k protein in this regulatory process will also be determined. The regulatory mechanisms to be defined in the studies proposed in this application will be of significance in both the field of vision biochemistry and the broader areas of receptor function and metabolic regulation.